Prion diseases are fatal protein misfolding neurodegenerative diseases (PMNDs) like Alzheimer, Parkinson, Huntington's Diseases or Amyotrophic Lateral Sclerosis. These diseases are due to the toxicity of a misfolded host protein. Available treatments are directed towards amelioration of symptoms and become ineffective as disease progresses. The lack of information concerning the exact mechanisms of neurodegeneration is a major hurdle to the development of disease- modifying treatments. With the aim to study these mechanisms, during the last 4 years, we developed a model for prion protein (PrP)-induced neuronal death. This model is based on the high, neuron-specific, toxicity of a monomeric misfolded form of recombinant PrP (TPrP); it reproduces morphological and molecular features of neuronal death occurring in mouse models of prion disease. We now set out to use this model to study the mechanism of neuronal death induced by misfolded PrP. We observed that TPrP enters neuronal cells, but not fibroblasts, and induces a profound NAD+ depletion via an ADP-ribosylation-dependent pathway. NAD+ depletion is ultimately responsible for neuronal death as NAD+ replenishment leads to full cellular recovery after TPrP-induced injury. The objective of this focused research proposal is to identify key effectors in the TPrP-induced toxicity pathway leading to NAD+ depletion and neuronal death. Our working hypothesis is that the knock-down of cellular factors critical to the execution of TPrP-induced death pathway will prevent neuronal death, and therefore these key players can be identified by implementing a genomic siRNA screen in our TPrP neurotoxicity assay. The proposed research will identify new therapeutic targets for prion protein-induced neurodegeneration thereby addressing a critical barrier to progress in the field of neuroprotective therapeutic interventions Because other PMNDs, like prion diseases, are due to the toxicity of a misfolded form of a host protein, and in view of the growing evidence for common mechanistic features, the results of the proposed research are likely to have a broad impact for the treatment of protein misfolding diseases. Subsequent projects will aim at using identified cellular factors to decipher the entire neuronal death pathway and developing new neuroprotective therapeutic strategies in prion diseases and possibly other protein misfolding neurodegenerative diseases.!